Lyme disease is the most common vector-borne disease in the United States, and its incidence and geographic distribution are expected to increase with further climate change. The agent is the spirochete Borrelia burgdorferi, which is transmitted by Ixodes scapularis ticks in the eastern U.S. A major reservoir for the infection is the mouse Peromyscus leucopus. Following the commercial withdrawal of the human Lyme disease vaccine, a plausible alternative for prevention of human disease is the targeting of a transmission-blocking vaccine to reservoirs of the infection, the proof-of-principle of which has been provided by this laboratory. However, further implementation of such measures is hindered by insufficient information about the immune responses of natural reservoirs for B. burgdorferi. In particular, it is not known whether the strain diversity of this pathogen can be attributed to positive selection by the reservoir's immune system. A reasonable candidate antigen for immune selection is the outer-surface protein C (OspC), which is expressed during early infection of mammals. The working hypothesis for this project is that the extensive diversity between OspC proteins of different strains of B. burgdorferi is the consequence of positive selection and that a major, if not sole, determinant of that selection is the immune response by reservoir hosts against OspC. Evidence in favor of this is the demonstration that antibody responses to OspC in natural and laboratory populations of white-footed mice are strain specific. The technical approach for this project (Aim 1) is to utilize the extensive database that we have developed on ospC genes and other B. burgdorferi loci for strains of the U.S. to develop (1) a protein microarray chip representing all known OspC types and other known antigens of B. burgdorferi, and (2) selected transgenic lines of B. burgdorferi that are isogenic except with respect of the ospC allele they express. These tools will be used in experiments of the two other specific aims. Aim 2. To immunize laboratory-reared P. leucopus mice with purified recombinant OspC proteins, following with an infectious challenge with B. burgdorferi of homologous or heterologous OspC type. We will examine the specificity of their anti-OspC immune responses on the OspC array and the ability of the mouse to allow or not establishment of borrelial infection after OspC pre-immunization, in an OspC type-specific way. Transgenic fl. burgdorferi will allow the isolation of the role of functional anti-OspC antibodies, and the effects of antibody binding on the spirochete. These experiments will demonstrate the degree of OspC type-specificity of the immune response, and the extent to which OspC type cross-reactivity determines the range of susceptibility of the host to different strains of B. burgdorferi. Aim 3. To extend these studies to the field by examining sera collected from wild P. leucopus for their reactivity against OspC and other antigens on the OspC protein array. The existing infections of the sampled mice will be determined by PCR of ear punch biopsies and sequencing of the ospC genes of PCR-positive mice. The functional or neutralizing immune responses of the P. leucopus will also be characterized with the transgenic B. burgdorferi constructs that are isogenic, except for of the OspC type to be tested.